U.S. patent number 6,986,841 [Application Number 10/674,652] was granted by the patent office on 2006-01-17 for fused-silica capillaries with photopolymer components.
Invention is credited to Jing-Ran Chen, Maria T. Dulay, Richard N. Zare.
United States Patent |
6,986,841 |
Zare , et al. |
January 17, 2006 |
Fused-silica capillaries with photopolymer components
Abstract
A separation column is provided with a photopolymer component
which, when irradiated, causes controlled porosity polymerization.
A particularly preferred embodiment is wherein the separation
medium is retained by a photopolymer frit, which can be reliably
and reproducibly generated with controlled porosity.
Inventors: |
Zare; Richard N. (Stanford,
CA), Dulay; Maria T. (Sunnyvale, CA), Chen; Jing-Ran
(Shaumburg, IL) |
Family
ID: |
24019792 |
Appl.
No.: |
10/674,652 |
Filed: |
September 29, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040055940 A1 |
Mar 25, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10008482 |
Nov 13, 2001 |
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09507707 |
Feb 18, 2000 |
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Current U.S.
Class: |
210/198.2;
210/635; 210/656; 96/101 |
Current CPC
Class: |
G01N
27/44704 (20130101); G01N 27/44747 (20130101); G01N
30/603 (20130101); G01N 30/6004 (20130101); G01N
2030/285 (20130101); G01N 2030/562 (20130101) |
Current International
Class: |
B01D
15/08 (20060101) |
Field of
Search: |
;210/635,656,198.2
;95/82,88 ;96/101 ;204/601,605,451,455 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 439 318 |
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Jul 1991 |
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EP |
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0 779 512 |
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Jun 1997 |
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EP |
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WO 98/29350 |
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Jul 1998 |
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WO |
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WO 99/30147 |
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Jun 1999 |
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WO |
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WO 00/49396 |
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Aug 2000 |
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WO |
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Primary Examiner: Therkorn; Ernest G.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation of application Ser. No. 10/008,482, filed
Nov. 13, 2001, now abandoned which is in turn a continuation of
application Ser. No. 09/507,707, filed Feb. 18, 2000, both
abandoned and both of which are incorporated herein in there
entireties by this reference.
Claims
What is claimed is:
1. A separation column comprising: a separation channel extending
between an inlet and an outlet and having a channel wall; and, a
separation medium in the channel, said channel having at least one
photopolymer frit adapted to retain the separation medium in the
channel, the at least one frit having a controlled porosity.
2. The separation column as in claim 1 wherein the photopolymer
frit is bound to an inner surface of the channel wall.
3. The column as in claim 1 wherein the photopolymer frit is
adjacent to the inlet, the outlet, or both.
4. The column as in claim 1 wherein the controlled porosity of the
photopolymer frit is a mean pore size of between about 1.0 .mu.m
and 5.0 .mu.m.
5. The column as in claim 1 wherein the separation channel is a
fused-silica capillary having an internal dimension in the range of
between about 5 and 300 .mu.m, and the photopolymer frit is of a
structure sufficient to withstand high pressure during packing of
the separation medium in the channel.
6. The separation column as in claim 5 wherein the photopolymer
frit is derived from a methacrylate monomer or a
methacrylate-substituted silicate.
7. The separation column as in claim 6 wherein the photopolymer
frit is derived from a methacrylate monomer polymerized via
photoinitiation.
8. The column as in claim 6 wherein the photopolymer frit is
derived from a photocurable methacrylate-substituted silicate.
9. The column of claim 1, said column having a first portion that
is filled with said separation medium and a second portion adjacent
to said first portion that transmits radiation.
10. The column of claim 9 wherein said second portion does not
contain said separation medium.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention generally relates to separation columns,
particularly capillaries useful in capillary zone electrophoresis,
including capillary electrochromatography, and more particularly to
separation columns that includes a photopolymer component. This
photopolymer component may be in the form of a frit.
2. Description of Related Art
Over the past decade, capillary zone electrophoresis (CZE), with
its high peak capacity (i.e., the number of peaks separated per
unit time), has developed into a powerful and widely used technique
for separating ionic species by their electrophoretic mobilities.
The lack of selectivity for uncharged analytes in CZE, however, has
remained more problematic. Several methods have been developed,
such as micellar electrokinetic chromatography (MEKC), to help
overcome this problem by providing a pseudostationary phase in
which uncharged compounds can be separated. The application of
methods such as MEKC is limited because of the restricted number of
pseudostationary phases that can be employed in this technique.
With the advent of capillary electrochromatography (CEC), where
both chromatographic and electrophoretic transport mechanisms are
combined, separation and analysis of mixtures of uncharged analytes
can be achieved using low sample volumes with high resolution and
efficiency. The increased interest in CEC for analytical
applications arises from the large plate numbers and relatively
high separation speeds achieved and the wide range of stationary
phases (those commonly used in high-performance liquid
chromatography) that can be used.
Many groups have reported on the use of slurry or electrokinetic
packing methods for the fabrication of electrochromatography
capillary columns with typical inner diameters of 75 .mu.m.
Reversed-phase capillary columns have been prepared, typically with
octadecyl silica (ODS) whose particle diameters are on the order of
1.5 to 5 .mu.m. In both packing techniques, the use of retaining
frits at both the inlet and the outlet of the capillary has been
required to prevent the chromatographic packing material from
exiting the capillary. Although systematic studies regarding the
effects of the frits on the performance of such capillaries have
not been reported, it is thought that these frits may degrade the
efficiencies of these capillary columns.
Alternative approaches have been reported for the preparation of
capillary columns that avoid the technical problems of frit
fabrication and column preparation associated with slurry and
electrokinetic packing. One approach uses bonded stationary phases.
Capillary columns prepared in this manner, however, suffer from low
retention and low sample capacities as well as long preparation
times. An alternative method for the preparation of open tubular
capillary columns uses monolithic packing technology. For example,
Dulay et al. have described the preparation and characterization of
monolithic porous capillary columns loaded with chromatographic
particles based on sol-gel chemistry (Dulay et al., Anal. Chem.,
70, pp. 5103-5107, 1998).
Nevertheless, where separation columns are desired with packing
material requiring frits, it would be desirable to have simple and
reproducible procedures for fabricating frits.
The conventional method of frit fabrication for a particle-packed
column involves thermal sintering of a section of the packing
material, such as ODS (octadecyl silica particles). This approach
has several disadvantages, including (1) difficulty in generating
the frit reliably and reproducibly, (2) alteration of the
characteristics of the stationary phase within the frit itself, (3)
difficulty in controlling the porosity of the frit, (4) weakness of
the capillary at the location of the frit, (5) band broadening
caused by the frit, (6) bubble formation and adsorption of polar
analytes on the frit. These problems can directly affect the column
performance and column-to-column reproducibility.
BRIEF SUMMARY OF THE INVENTION
In one aspect of the present invention, a separation column is
provided that comprises a separation channel having a channel wall
and a separation medium in the channel, and wherein the separation
column includes a photopolymer component. In one embodiment, the
photopolymer component is a frit adapted to retain the separation
medium. The frit has a controlled porosity and is preferably
derived either from a methacrylate monomer that is polymerized via
photoinitiation or from a methacrylate-substituted silicate that is
photocurable. Because polymerization is initiated or cured by means
of radiation, the position of the polymeric component can be
localized and the porosity reproducibly controlled. Further,
polymerization can be accomplished without removing the typical
protective polyimide coating.
Another aspect of the invention is a method of fabricating frits in
fused-silica capillaries, and more particularly in fabricating
controlled porosity frits. Advantages of the invention include (i)
easy and rapid preparation, (i) short reaction times, (iii) UV
transparency of the photopolymer, and (iv) fine control of pore
sizes. This results in a short total frit preparation time, and
avoids the use of elevated temperatures.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1, panels (A), (B), and (C) are SEM micrographs where panel
(A) is an oblique view of a photopolymer outlet frit in a capillary
(with no particles present), panel (B) is a 5.times. magnified view
of (A), and panel (C) is a cross-section of a photopolymer inlet
frit with embedded chromatographic particles;
FIG. 2 is a graphical representation showing in panel (A) a plot of
absorbance versus retention time for one column and panel (B) for
another column where the two analytes were thiourea and
2-methyl-naphthalene (in order of elution);
FIG. 3 are three representative electrochromatograms using an
embodiment of the invention (column 2 of FIG. 2, panel (B)), where
the analytes were thiourea, benzyl alcohol, benzaldehyde, and
2-methyl-naphthalene (in the order of elution); and
FIG. 4 (panels A and B) are two representative
electro-chromatograms where in panel A Taxol (a promising
anti-cancer drug) was run, whereas panel B was a Taxol analog
(Baccatin D), both in relatively large-bore capillaries having
frits of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Capillary zone electrophoresis (CZE) has long been proven to be an
attractive analytical technique for separating ionic species by
their electrophoretic mobilities. The limitation of CZE, however,
has been its ineffectiveness in separating neutral compounds. To
this end, capillary electrochromatography (CEC), which combines
both CZE and chromatography, has been developed to improve the
separation of neutral species. A combination of mechanisms based
partitioning and electrophoretic mobility can be used to describe
the separation that occurs in CEC.
An existing technique for the preparation of retaining frits in
packed capillary columns involves the sintering of silica beads
within the columns. This sintering method is the common technique
presently employed for the preparation of capillary column frits.
Disadvantages of the sintered silica frits include the
irreproducibility of the frit porosity and length and the need for
very high temperatures.
We have used polymer photochemistry to prepare photopolymer frits
in capillary columns. The photopolymer method has several
advantages over the existing sintered silica methods, including (i)
easy and rapid preparation, (ii) short reaction times at room
temperature, (iii) UV transparency of the photopolymer, (iv) fine
control of pore sizes, and (v) control of frit lengths and frit
position.
In one embodiment of the present invention, photopolymer frits are
prepared from a mixture of methacrylate monomers, porogenic
solvents, and a photo initiator. Methacrylate monomers are known
for use in the preparation of chromatographic separation media
(Viklund et al., Chemistry of Materials, 9, pp. 463-471, 1997).
Viklund et al. studied a variety of polymers prepared by
photopolymerization and found them suitable as sorbants in
analytical chemistry, and Viklund et al. is incorporated herein by
reference in its entirety.
One aspect of our research concentrated on the preparation of the
suitable frits and their effects on CEC separations. We chose a
mixture of isooctane and toluene as a particularly preferred
porogenic solvent for the preparation of porous polymers based on
methacrylate. The pore sizes in the polymer mixture may be
controlled through the use of porogenic solvents and by variations
in the molar ratios of the monomers and the porogens. For example,
by varying the proportions of these solvents, frit pore sizes of
between about 1.0 and 5.0 microns, more preferably 2.5 and 4.0
.mu.m, have been used in our experiments. The relationship of the
component ratios and pore sizes has been discussed, for example, by
Viklund et al., p. 464. Photopolymerization can be achieved even in
silica capillaries without removal of the polyimide coating. This
procedure is particularly advantageous for the preparation of inlet
frits. It was found that, by the hydrolysis of glycidyl
methacrylate, the hydropholicity of the frit could be increased.
This minimized the retaining effect of frits on the columns with
reversed-phase chromatographic materials.
In another embodiment of the present invention, photopolymer frits
are prepared from photocuring a methacrylate-substituted silicate,
via a sol-gel reaction. Suitable photocured sol-gels are known and
useful for practicing this aspect of the invention, such as
described by Etienne et al., J. Sol-Gel Sci. & Tech., 13, pp.
523-527 (1998), which is incorporated in its entirety by reference.
Briefly, a monomer such as 3-(trimethoxysilyl) propyl methacrylate
(MAPTMS) is irradiated to form a sol-gel matrix. Such a reagent is
similar to the methacrylate-based reagent already described in the
first embodiment of the present invention, and is photocurable.
Broadly, the metal alkoxide sol-gel process is described by Brinker
et al., Sol-Gel Science, Academic Press, Inc., New York (1990).
When the gel is cured, a hard porous glass is obtained.
It is a common practice to remove the polyimide coating by chemical
or thermal means in the detection area of the capillary because the
polyimide allows for only a small transmittance of UV light into
the capillary. In the preparation of frits in packed capillary
columns used in CEC, the high heat required to center silica beads
in the capillary columns leads to the removal of the polyimide
coating, which imposes the fragile fused-silica at the portion of
the capillary that contains the silica frit. However, in practicing
the invention through use of photopolymers as components (such as
for an inlet and outlet frit or as the supporting matrix itself),
there is no need to remove the polyimide coating since the
photochemical used to prepare the polymers can be conducted at
relatively low levels of UV light. Thus, the capillary outer
surface coatings remain intact and the mechanical strength of the
column is maintained.
Images of a photopolymer frit embodiment based on the
polymerization of methacrylate monomers with and without ODS
particles are illustrated by the electron micrographs shown in FIG.
1, panels (A)-(C). FIG. 1, panel (A) shows the photopolymer in a 75
.mu.m i.d. capillary, and FIG. 1, panel (B) provides a magnified
view of the polymer. FIG. 1, panel (C) shows the polymer structure
in the presence of 1.5 .mu.m ODS particles. This micrograph also
demonstrates that the photopolymer loses its spherical shape in the
presence of the ODS particles and the pores entrap the silica beads
and hold them within their domains.
After the polymerization had been completed, buffer solution was
flushed (using a hand-held manual syringe pump) through a capillary
column with one frit and no ODS particles present. There was almost
no increase in the back-pressure observed as compared with the
empty capillary without a photopolymer frit. This observation
indicates that the frit does not significantly increase the flow
resistance of the system. Although no specific functionalization of
the inner capillary surface preceding the polymerization was
carried out, the outlet frit easily withstands a short exposure of
a high pressure (>6000 psi) used during the column packing. This
behavior demonstrates that the monolithic porous polymer frit is
strongly bound to the inner surface of the bare capillary wall. No
particles of the packing were found to pass through the frit pores
during either the packing procedure or the CEC applications,
despite their size, which is smaller than that of the mean pore
size. This results from the cooperative action of several effects
such as the tortuous paths of the pores and the presence of
bottleneck-shaped pores within the monolithic structure. Five
columns were prepared with this packing procedure and, in all
cases, the photopolymer frits exhibited similar mechanical
stabilities under our experimental conditions.
In contrast to some other procedures, the UV photoinitiated
polymerization does not require elevated temperature for the
reaction to be completed. Therefore, the mobile phase used for
packing remains in both the inlet frit and the packing during
polymerization. Consequently, the conditioning time for the column
prior to its use is shortened significantly.
No bubble formation occurred during all the separation runs using
1.5 .mu.m ODS packed capillary columns with photopolymer frits
based on methacrylate polymerization. The suppression of bubble
formation might be due to both the addition of SDS in the running
buffer and the replacement of sintered-silica frits with
photopolymer frits.
The efficiency, N, the plate height, H, and the reduced plate
height, h, of a packed column (column 2) embodiment with
photopolymer frits based on the polymerization of methacrylate
monomers are listed in Table 1.
TABLE-US-00001 TABLE 1 Column Efficiency, Plate Height, and Reduced
Plate Height of Column 2* Analyte N/m H (.mu.m) h (dimensionless)
thiourea 220 000 4.5 3.0 2-methylnaphthalene 205 000 4.9 3.3
*Column 2: 75-.mu.m i.d. .times. 27-cm (20 cm packed with 1.5-.mu.m
ODS particles). Mobile phase: 20% (v/v) 5 mM phosphate with 2 mM
SDS (pH 7.0) and 80% (v/v) acetonitrile.
FIG. 2 compares the separation of two neutral compounds, thiourea
and 2-methylnaphthalene, achieved in columns furnished with porous
polymer frits characterized by mean pore diameters of 2.5 (FIG. 2,
panel (A)) and 4.0 .mu.m (FIG. 2, panel (B)), respectively. The
column with 4-.mu.m frits exhibits shorter retention times and a
better column efficiency compared with those of the other
column.
A systematic study of the run-to-run reproducibility for the
analysis of a mixture of neutral molecules consisting of thiourea,
benzyl alcohol, benzaldehyde, and 2-methylnapththalene was carried
out over a period of 3 days. FIG. 3 shows electrochromatograms of
runs 10, 30, and 50 of the Table 1 embodiment. There is almost no
variation in retention times of all test compounds. Table 2 shows
the relative standard deviations of the capacity factor, k.sup.1,
the efficiency, N, and the resolution, R, for each compound. These
RSDs for all three monitored variables, averaged over 60 runs, were
3.5%, 3.3%, and 5.5%, respectively.
TABLE-US-00002 TABLE 2 Relative Standard Deviation of Capacity
Factor (k.sup.1), Column Efficiency (N), and Resolution (R.sub.1)
over 80 Separations for Column 2* Relative Standard Deviation (%)
Analyte k.sup.1 N R.sub.5 thiourea 2.0 3.0 benzyl alcohol 3.2 5.5
3.2 benzaldehyde 2.1 5.3 2.7 2-methylnaphthalene 2.6 3.6 2.8
*Column 2: 75-.mu.m i.d. .times. 27-cm (20 cm packed with 1.5-.mu.m
ODS particles). Mobile phase: 20% (v/v) 5 mM phosphate with 2 mM
SDS (pH 7.0) and 80% (v/v) acetonitrile.
The integrity of the packed column remained unchanged, and the
column is believed to be useful for a much longer period of time.
In FIG. 3, the efficiencies of thiourea, benzyl alcohol,
benzaldehyde, and 2-methylnaphthalene are 200 000 plates/m, 160 000
plates/m, 60 000 plates/m, and 20 000 plates/m, respectively. The
unretained compound, thiourea, gave the highest efficiency. The
efficiencies dropped for the compounds that were more retained than
thiourea. The significant efficiencies drop of the last two peaks
compared with those of FIG. 2 are mainly due to the use of a lower
percentage (50%) of acetonitrile in the running buffer.
Furthermore, the peak for 2-methylnaphthalene shows tailing that is
mainly due to its adsorption to the surface of the ODS
particles.
Experimental
Materials. The monomers trimethylolpropane trimethacrylate (TRIM)
and 2,3-epoxypropyl methacrylate (glycidyl methacrylate, GMA) were
of the highest purity available from Aldrich (Milwaukee, Wis.).
Toluene and 2,2,4-trimethylpentane (isooctane) from Sigma (St.
Louis, Mo.) were used as porogenic solvents. The fused capillaries
used in this study were purchased from Polymicro Technologies
(Phoenix, Ariz.). The 1.5 .mu.m spherical ODS particles were
provided by Micra Scientific, Inc. (Lafayette, Ind.),
.alpha.-Methoxy-.alpha.-phenylactophenone (benzoin methyl ether,
99%), thiourea, benzaldehyde, benzyl alcohol, 2-methylnaphthalene,
sodium phosphate, and acetonitrile (HPLC grade) were purchased from
Aldrich (Milwaukee, Wis.). Water was purified with an Ultrapure
water system from millipore (Milford, Mass.).
Polymerization Procedure. The photopolymerization procedure was
carried out as previously described (Viklund et al., Chem. Mater.,
9, pp. 463-471, 1997). In situ free-radical polymerization was
initiated by irradiating the monomer solution in 4 mm i.d. quartz
tubes and 75 .mu.m i.d. capillaries for 60 minutes at room
temperature in a Spectrolinker XL 1500A (Spectronics Corp.,
Westbury, N.Y.) with six 15 W fluorescent blacklight tubes,
producing UV light of predominantly 365 .mu.m(?) wavelength. After
one hour, the porogen, unreacted monomers, and other soluble
compounds were removed from the pores by washing with ethanol using
an HPLC pump.
Pore-Size Measurement. The pore-size distribution of the porous
polymer was determined for the samples prepared in the 4 mm tubes
from the same polymerization mixtures as the capillaries, by
mercury intrusion porosimetry using an automated custom-made
combined BET sorptometer-porosimeter (Porous Materials, Inc.,
Ithaca, N.Y.).
Frit Fabrication and Column Packing. An outlet frit was prepared by
introducing the monomer mixture into the capillary. The two ends of
the capillary were sealed with Parafilm. The capillary was then
covered by aluminum foil, leaving 1 mm of the outlet section
without polyimide coating exposed to the UV light. The rest of the
outlet section that was masked during photopolymerization because
the detection window for CE experiments. After an hour of
polymerization at room temperature, the unreacted monomer solution
was flushed from the column by a syringe pump. After slurry packing
the column, 1.5 .mu.m ODS particles were filled up to the inlet end
of the capillary. The same procedure to create the outlet frit was
employed to form the inlet frit. The resulting columns were
preconditioned with the mobile phase by pressurizing the column
inlet to approximately 500 psi with a manual syringe pump (Unimicro
Technologies, Inc., Pleasanton, Calif.) for a few hours prior to
their use. It was noticed that the polymerization also took place
without removing the polyimide coating, but the process took about
four to six hours.
Capillary Electrochromatography. The CEC experiments were performed
with a Beckman model 2000 P/ACE capillary electrophoresis system
Beckman Coulter, Fullerton, Calif.) equipped with an UV absorbance
detector. The 75 .mu.m i.d. packed capillary column was installed
in the cartridge holder that was then inserted into the instrument.
There is no pressure applied to the packed column in this system.
Once the packed column was installed, it was further conditioned by
driving the mobile phase through the capillary at an applied
voltage of 5 kV for 1 hour prior to use. Samples were introduced
electrokinetically at the anodic end of the capillary column. The
mobile phase employed in these separations was a 5 mM phosphate and
2 mM SDS buffer (pH 7.0) containing 80% (v/v) acetonitrile.
Separations were performed at an applied voltage of 10 kV and at a
temperature of 20.degree. C. The analytes were detected by
monitoring their absorbance at 254 nm.
A mixture of neutral compounds was separated on two columns, 1 and
2. Column 1 was a 75 .mu.m i.d..times.27 cm (20-cm packed with 1.5
.mu.m ODS particles fused-silica capillary. The frit pore size of
this column was 2.5 .mu.m. Column 2 was the same as column 1,
except the frit pore size was 4.0 .mu.m.
Semi-preparative CEC work was carried out using home-made CE
instrument. The capillary column (550 .mu.m i.d. and 650 .mu.m
o.d.) was packed with 1.5 .mu.m diameter ODS particles using slurry
packing method. The frits of these large-bore capillary columns
were prepared from a mixture of methacrylate monomers, porogenic
solvents, and a photo initiator (Vildund et al.) in the same manner
as already described. The running buffer was a mixture of 20% 5 mM
phosphate and 80% acetonitrile. The total length of the capillary
was 34 cm. The applied voltage across the CEC column was 2 kV. The
sample injection volume was 150 nl. Electrochromatograms of Taxol
and Baccatin III analysis, respectively, are shown by panels A and
B of FIG. 4.
It is to be understood that while the invention has been described
above in conjunction with preferred specific embodiments, the
description and examples are intended to illustrate and not limit
the scope of the invention, which is defined by the scope of the
appended claims.
* * * * *